Plasma fractionation and product therefrom



2,47,348 Patented Aug. 12, 1958 PLASMA FRACTHUNATION AND PRODUCTTHEREFRGM Heron 0. Singher, Plainiield, and Emanuel A. Swart,Somerville, N. J., assignors to Ortho lharmaceuticai Corporation, acorporation of New Jersey No Drawing. Application May 27, 1954 SerialNo. 432,895

4 Claims. (Cl. 167-74) This invention relates to a method for thepreparation and isolation of a substance from horse, bovine, human orrabbit plasma capable of increasing the activity of thromboplasticmaterial. More specifically, the invention relates to the fractionationof horse or rabbit plasma whereby there is obtained a substance havingthe ability to increase the activity of a thromboplastic material whichhas a prothrombin time, as determined by the Shapiroeiner method, longerthan nine seconds.

Thromboplastin has accepted value for use in the determination ofprothrombin time, which is a measure of the amount of prothrombinpresent in a tested blood sample. The determination of prothrombin timeis useful clinically, for the fact that it varies with a variety ofclinical situations has been well established. It is known that vitamindeficient diets may result in prolonged prothrombin time. Biliarydiseases frequently result in prolonged prothrombin time and areconsidered to be related to impaired vitamin K absorption. Impairedliver function results in prolongation of prothrombin time. A variety ofdrugs such as the salicylates and especially Dicumarol affect theprothrombin time to a degree considered sufficient to be of clinicalsignificance.

The two-stage theory of Morawitz for the mechanism of blood coagulationpostulates, as a first stage, the interaction of prothrombin, calciumion, and thromboplastin xhich results in the formation of thrombin and,as a secnd stage, the reaction of thrombin with fibrinogen to formfibrin. Fibrin fibers are largely responsible for the characteristicproperties of clotted blood. It has been shown that the addition toblood of small amounts of thromboplastin can accelerate clotting time,generally referred to as prothrombin time, fromthe usual several minutesdown to a few seconds. Thromboplastin, otherwise known as theplatelet-tissue factor, is essential to the blood clotting mechanism buthas not been definitely identified chemical-1y. The mechanism of theactivity and function of thromboplastin in blood clotting is not settledbut most workers believe it to be enzymatic, and that thromboplastinacts to catalyze the conversion of prothrombin to thrombin probablythrough an intermediate prothrombin thromboplastin calcium complex.Since thrombin is a protein essential to the formation of fibrin andthromboplastin is necessary for the conversion of prothrombin tothrombin, the measurement ofprothrombin time, wherein a standardizedpreparation of thromboplastin is used, has come to be considered asyielding information of great clinical. value. a

It is an object of this invention to provide a process for thefractionation of horse, bovine, human, or rabbit plasma whereby asubstance is isolated which is capable of increasing the activity ofthromboplas'tic material which has a prothrombin time of longer thannine seconds.

it is another and further object of this invention to provide asubstancefrom horse,.bovine,, human, ,or rabbit plasma which upon additiontothromboplastic material obtained from rabbitbrain or lung tissue, or amixture thereof, results in a decrease in the prothrombin time of plasmaas determined by our modification of the Shapiro- Weiner method.

Other objects and more particular advantages of the invention willappear from the following description as well as the appended claims.

.The objects of this invention are accomplished by a process in whichde-prothrombinized horse, bovine, human, or rabbit plasma issubstantially freed of albumin and alphalobulins by extraction with afirst buffered aqueous alcohol solution and extracted with a secondbuffered aqueous alcohol solution to obtain a substance which, uponaddition to thromboplastic material having a prothrombin time of longerthan nine seconds, increases the activity thereof.

More particularly, in the process of this invention, horse, bovine,human, or rabbit plasma to which a heparin sodium solution has beenadded to prevent coagulation, is centrifuged in order to obtain theplasma. The plasma is slurried with two to five percent, and preferablyfive percent, of barium sulfate, the barium sulfate being expressed ingrams, and the plasma in cubic centimeters. At least two percent ofbarium sulfate is required toremove all the prothrombin, and thepresence of more than five percent results in a mixture of such aconsistency that physical handling thereof is extremely difficult. Theslurry is stirred for one hour in order that the maximum amount ofprothrombin be completely adsorbed, and is then centrifuged. It ispreferred that the adsorption be repeated to insure complete removal ofprothrombin, preferably with the same amount of barium sulfate, and uponcentrifugation after the second adsorption, the plasma is prothrombinfree. Albumin and alpha-globulinsare extracted from the prothrombimfreeplasma by the addition of a first buffered aqueous alcohol solution andcentrifugation. The alcohol may be ethanol, methanol or a mixture ofmethanol and ethanol and may be present in an amount of from about 15 to30 percent by volume, but it is preferred that the amount be about 25percent. If the amount is less than about 15 percent, a significantamount of active substances remain in solution, and if the amount isgreater than about 30 percent a substantial amount of inactive materialis present in the precipitate. The first aqueous alcohol solution isbuffered Within the pH range of from 4 to 5, the preferred pH being 4.2to 4.6. Buffering below a pH of 4 results in incomplete solution ofalbumin in the extracting solution and buffering above a pH of 5 resultsin a significant amount of the desired plasma fraction being lost in theextracting solution. In general, any buffer system capable ofmaintaining the pH of the mixture of the first aqueous alcohol solutionand prothrombin-free plasma within the range of 4 to 5 may be used. Thepreferred buffer system is sodium acetate-acetic acid; however, buffersystems such as sodium succinate-succinic acid, and sodium acidphthalate-phthalic acid, have been found suitable.

The volume of first aqueous alcohol solution used may vary Widely, butthe most efiicient removal of albumin and alpha-globulin from theprothrombin-free plasma is accomplished when the volume is three to fivetimes the volume of the plasma. It is preferred that the volume of thefirst aqueous alcohol solution be about four times the volume of theplasma.

It is necessary that the extraction and removal by centrifugation ofalbumin and alpha-globulins in solution in the first aqueous alcoholsolution be accomplished at a low temperature in order that they beefiiciently removed from the plasma. Extraction and removal at a highertemperature results in denaturation of albumin and alpha-globulins bythe alcohol and incomplete removal thereof in the extracting solution.The pro thrombin-free horse, bovine, human, or rabbit plasma is cooledto 5 to 0 C., and preferably to 0 CL, and

the first aqueous alcohol solution, which has been cooled to 5 to l C.,is slowly added with stirring and during the course of the addition, thetemperature of the mixture is maintained between 0 and C., andpreferably at about 5 C. After addition is completed the mixture isstirred for about 30 minutes and during this time the temperature of themixture is maintained at 0 to -5 C., and preferably at 5 C. Immediatelyafter stirring is discontinued, the mixture is centrifuged and duringthe centrifugation the temperature is maintained at 0 to 5 C., andpreferably 5 C. The supernatant, which consists mainly of albumin andalphaglobulins is discarded and the residue is extracted with a secondbuffered aqueous alcohol solution to obtain the desired plasma fraction.

The alcohol in the second aqueous alcohol solution may be ethanol,methanol, or a mixture of ethanol and methanol and the alcohol, ormixture of alcohols, is present in an amount from 5 to 20 percent byvolume and preferably in an amount of about 16 percent by volume. Thesecond aqueous alcohol solution is bufiered at a pH within the range offrom 6 to 8, and preferably 6.8 to 7.2. A solution buffered outside thispH range extracts significantly less of the desired plasma fraction fromthe residue of the first extraction. The second aqueous alcohol solutioncontains alkali metal salt of an amino acid having not more than sixcarbon atoms, such as alanine, glycine, proline, or serine. In general,other amino acids are not sufficiently soluble in the second extractingsolution. The alkali metal salt acts as a stabilizing agent for thematerial to be extracted from the plasma, as a solublizer for betaandgamma-globulins, and as an active part of the buffer system. The aminoacid is present in an amount within the range of 4 to 6 percent byweight, and preferably 5.4 to 5.8 percent by weight. At a concentrationless than 4 percent by weight, the solubilizing effect o-fthe amino acidis significantly decreased; on the other hand, the solubilizing efiectis not increased when the concentration is greater than 6 percent.Phosphates such as monoor di-sodium phosphate are also present in thesecond aqueous alcohol solution as part of the buffer system and the pHof the solution is adjusted to the desired value with an alkali metalhydroxide. Any buffer system may be used which is effective within a pHrange of 6 to 8.

The amount of the second aqueous alcohol solution used in the extractionof the albumin and alpha-globulinfree residue may vary widely but anamount of solution three to five times, and preferably four times, thevolume of the original plasma results in the most efficient recovery ofthe desired plasma fraction. The second aqueous alcohol solution ispreferably cooled to 0 to 5 C. and added at that temperature to theresidue. The mixture of residue and second aqueous alcohol solution isstirred at a temperature of 0 to 5 C., and preferably at 5 C., for about30 minutes, and then centrifuged and the residue from the centrifugationwhich consists mainly of beta-globulins and fibrinogen is discarded.During centrifugation the temperature is preferably kept at -1 to 5 C.The supernatant, which contains the desired plasma fraction, isfiltered, dialyzed against distilled water at a temperature of l to 5C., and the dialystate is lyophilized. (The term dialysate as used inthis specification designates the material which has failed to passthrough the dialysis membrane.) Denaturation of the desired plasmafraction is at a minimum when the temperature during dialysis is withinthe range of l to 5 C. The solid material obtained upon addition tothromboplastic material enhances its thromboplastic activity.

In order that those skilled in the art may better understand how thepresent invention may be carried into effect, the following examples aregiven by way of illustration but not by way of limitation.

Example I Fifty ml. of heparin sodium solution were added to 20 litersof horse blood and the mixture was centrifuged for 30 minutes at 25 C.Nine liters of the supernatant plasma were slurried with 450 grams ofbarium sulfate at 25 C., stirred for one hour and centrifuged. Thesupernatant was slurried with 450 grams of barium sulfate, stirred forone hour at 25 C. and centrifuged. The prothrombin-free plasma obtained,which had a volume of eight and one-half liters, was cooled to 0 C.

Thirty-four liters of an aqueous alcohol solution buffered at a pH of4.6 was cooled to a temperature of 0 C. and added to the plasma at sucha rate that the temperature of the mixture throughout the addition wasbelow 0 C. The mixture was stirred for 30 minutes at 5 C. after additionwas complete and then centrifuged and the temperature of the mixture wasmaintained at a temperature of 5 during the centrifugation. Thesupernatant, which contained substantially all the albumin andalpha-globulins of the plasma, was discarded. The

aqueous alcohol solution contained per liter, 12 /2 ml.

of methanol, 237 /2 ml. of percent ethanol, and 0.6 ml. of an acetatebuffer solution prepared by diluting a mixture of 20 ml. of 4 molarsoduim acetate and 40 ml. of 10 molar acetic acid to ml. with water. Theresidue from the centrifugation was finely dispersed with a spatula in34 liters of the aqueous alcohol extracting solution buffered at a pH of7. The temperature of the extracting solution and residue during thedispersion was maintained at 5 C. and after dispersion was complete themixture was stirred for 30 minutes at a temperature of 5 C. andcentrifuged. The temperature of the mixture during the centrifugationwas maintained at S C. The supernatant was filtered and dialyzed againstdistilled water. The temperature of the supernatant during filtrationand dialysis was maintained at 5 C. The dialysate was lyophilized and350 grams of solid were obtained. The solid had no demonstrablethromboplastic activity. The aqueous alcohol extracting solutioncontained, per liter, 8 ml. of methanol, 152 ml. of 95 percent ethanol,56 grams of glycine, 3.12 ml. of a solution of sodium glycinate,prepared by dissolving 4.5 grams of glycine and 2.0 grams of sodiumhydroxide in 100 ml. of water, 4 ml. of 0.5 molar disodium hydrogenphosphate, and 2.76 ml. of 0.5 molar monosodium dihydrogen phosphate.

Example II Three ml. of heparin sodium solution were added to 1200 ml.of rabbit blood and the mixture was centrifuged for 30 minutes at 25 C.Six hundred-twenty ml. of the supernatant plasma were slurried with 31grams of barium sulfate at 25 C., stirred for one hour and centrifuged.The supernatant was slurried with 31 grams of barium sulfate, stirredfor one hour at 25 C., and centrifuged. The prothrombin-free plasmaobtained, which had a volume of 656 ml. was cooled to 0 C. Two thousandtwo hundred-sixty ml. of an aqueous alcohol solution buffered at a pH of4.6 was cooled to a temperature of 0 C. and added to the plasma at sucha rate that the temperature of the mixture throughout the addition wasbelow 0 for 30 minutes at -5 C. after addition was complete and thencentrifuged and the temperature of the mixtur was maintained at atemperature of 5 during the cen trif-ugation. The supernatant, whichcontained substantially all the albumin and alpha-globulins of theplasma,

was discarded. The aqueous alcohol solution contained per cent ethanoland 0.6 ml. of an acetate buffer solution prepared by diluting a mixtureof 20 ml. of 4 molar sodium acetate and 40 ml. of 10 molar acetic acidto 100 ml. with water. The residue from the centrifugation was finelydispersed with a spatula in 2260 ml. of the aqueous C. The mixture wasstirred liter, 12% ml. of methanol, 237 /2 ml. of 95 peralcoholextracting solution buffered at a pH of 7. The temperature of theextracting solution and residue during the dispersion was maintainel at,5 C. and after dispersion was complete the mixture was stirred for 30minutes at a temperature of 5 C. and centrifuged. The temperature of themixture during the centrifugation was maintained at -5 C. Thesupernatant was filtered and dialyzed against distilled water. Thetemperature of the supernatant during filtration and dialysis wasmaintained at 5 C. The dialysate was lyophilized and eight grams ofsolid were obtained. The solid had no demonstrable thromboplasticactivity. The aqueous alcohol extracting solution contained, per liter,8 ml. of methanol, 152 ml. of 95 percent ethanol, 56 grams of glycine,3.12 ml. of a solution of sodium glycinate, prepared by dissolving 4.5grams of glycine and 2.0 grams of sodium hydroxide in 100 ml. of water,4 ml. of 0.5 molar disodium hydrogen phosphate, and 2.76 ml. of 0.5molar monosodium dihydrogen phosphate.

A plasma fraction prepared as above has the ability to increase theactivity of a thromboplastic material which has a prothrombin time, asdetermined by our modification of the Shapiro-Weiner method, longer thannine seconds. A suitable thromboplastic material was prepared asfollows:

Seventy-six grams of frozen rabbit brain and 1440 grams of frozen rabbitlung were homogenized at 5 C. for one minute in the presence of 7600 ml.of an aqueous solution containing,,per liter, 150 ml. of an alcoholicsolution prepared by adding 7.5 ml. of methanol, grams of glycine, 4.8ml. of one molar aqueous sodium acetate solution, and 2.6 ml. of onemolar aqueous acetic acid solution to 142.5 ml. of 95 percent ethanol.The homogenate was stirred for two hours at 5 C. and centrifuged at 5 C.for thirty minutes. The supernatant liquid was filtered, dialyzedagainst distilled water at 5 C. and lyophilized. Forty-four andforty-six hundredths grams of thromboplastic solid material wereobtained.

The thromboplastic activity, as measured by the prothrombin time, of thethromboplastic material prepared as above Was determined as follows:

A calcium-thromboplastin suspension was prepared in a test tube byadding 100 milligrams of lyophilized solid to ml. of 0.85 percentaqueous sodium chloride solution, admixing by inverting the tube threeor four times until a uniform suspension was obtained, keeping thesuspension in a water bath at 4650 C. for twenty minutes, centrifuging,cooling the supernatant to room temperature, adding 0.1 ml. of 0.25molar calcium chloride 50- lution to 4 ml. of the suspension, mixing asabove, and centrifuging again. Two-tenths ml. of the slightly turbidsupernatant liquid was added to 0.1 ml. of fresh, oxalated human plasmawhich had been prepared by the addition of 0.1 molar aqueous sodiumoxalate solution to fresh human blood in the proportion of one partsodium oxalate solution to nine parts of blood and centrifugation of theoxalated blood. The mixture of supernatant solution and oxalated humanplasma was agitated at 37 C. by tilting the test tube back and forth andtiming the first appearance of a fibrin clot. Clot formation wasdetected in twenty-five seconds after addition of the supernatant liquidto human plasma.

The calcium-thromboplastin suspension, in the form of the slightlyturbid supernatant liquid prepared above, was mixed with a solution ofthe plasma fraction, prepared according to Example II and containingfive mg. of plasma fraction per ml. of 0.85 percent aqueous sodiumchloride solution, so that a series of mixed solutions containingvarying proportions of the calcium-thromboplastin suspension and thesaline solution of the plasma fraction resulted. The first and secondcolumns in the table below give the amounts in milliliters ofcalciumthromboplastin suspension and saline solution of the plasmafraction, respectively, in the series of mixed solutions. Thethromboplastic activity, as measured by prothrombin time, of each of thesolutions of the series was determined by adding 0.2 ml. of the solutionto 0.1 ml. of fresh, oxalated human plasma which had been prepared bythe addition of 0.1 molar aqueous sodium oxalate solution to fresh humanblood in the proportion of one part sodium oxalate solution to nineparts of blood and centrifugation of the oxalated blood. The mixture ofsolution and oxalated human plasma was agitated at 37 C. by tilting thetest tube back and forth and timing the first appearance of a fibrinclot. The values in the third column of the table below represent thetime in seconds for clot formation to occur following addition of thesolution to the human plasma.

All determinations of thromboplastic activity, as measured byprothrombin time, were determined by our modification of theShapiro-Weiner method for determining prothrombin time of blood, whichis described in a book entitled: Coagulation, Thrombosis and Dicumarol,by Shapiro and Weiner, published in 1949 by the Brooklyn Medical Press,Brooklyn, New York.

It will be obvious to those skilled in the art that various changes maybe made without departing from the spirit of the invention and thereforeit is to be understood that the invention is not limited to what isdescribed in the specification and examples but only as indicated in theappended claims.

What is claimed is:

1. A process for the preparation of a substance capab of increasing theactivity of thromboplastic material, con-- ducted at a low temperaturethroughout, comprising the steps: adding to a unit volume ofprothrombin-free plasma selected from the class consisting of horse,bovine, human, and rabbit prothrombin-free plasma, three to five volumesof a first aqueous alcohol solution buffered at a pH of 4-5 andcontaining about l530 percent by volume of an alcohol selected from theclass consisting of methanol and ethanol and mixtures thereof, stirringand centrifuging the mixture, separating the supernatant from theresidue; and adding to the residue three to five volumes based on plasmavolume of a second aqueous alcohol solution bufiered at a pH of 6-8 andcontaining 5-20 percent by volume of an alcohol selected from the classconsisting of methanol and ethanol and mixtures thereof and 4-6 percentby weight of an alkali metal salt of an amino acid having not more thansix carbon atoms, stirring and centrifuging the mixture, filtering thesupernatant, dialyzing the filtered supernatant against distilled water,and lyophilizing the dialyzed supernatant.

2. A process for the preparation of a substance capable of increasingthe activity of thromboplastic material comprising the steps: adding ata temperature of 5 to 10 C. to a unit volume of prothrombin-free plasmaselected from the class consisting of horse, bovine, human, and rabbitprothrombin-free plasma at a temperature of 5 to 0 C., about fourvolumes of a first aqueous alcohol solution buttered at a pH of 4.24.6and containing about 25 percent by volume of an alcohol selected fromthe class consisting of methanol and ethanol and mixtures thereof,stirring and centrifuging the mixture, and separating the supernatantfrom the residue while the temperature is maintained at 0 to 5 C. andadding while the temperature of 0 to 5 C. to the residue at atemperature is 0 to 5 C., three to five volumes based on plasma volumeof a second aqueous alcohol solution, at a temperature of 0 to 5 C.,buifered at a pH of 6.8 to 7.2 and containing about 16 percent by volumeof an alcohol selected from the class consisting of methanol and ethanoland mixtures thereof and 5.4-5.8 percent by weight of an alkali metalsalt of an amino acid having not more than six carbon atoms, stirringthe mixture while the temperature is at to C., centrifuging the mixture,filtering the supernatant, and dialyzing the filtered supernatantagainst distilled water while the temperature is at 1 to 5 C., andlyophilizing the dialyzed supernatant.

3. A substance capable of increasing the activity of thromboplasticmaterial prepared at a low temperature from prothrombin-free plasma,selected from the class consisting of horse, bovine, human, and rabbitprothrombin-free plasma, by adding to a unit volume of the plasma 3-5volumes of a first aqueous alcohol solution buffered at a pH of 4-5 andcontaining about -30 percent by volume of an alcohol selected from theclass consisting of methanol and ethanol and mixtures thereof, stirringand subsequently centrifuging the mixtures, separating the supernatantfrom the residue; and adding to the residue 3-5 volumes based on plasmavolume of a second aqueous alcohol solution buffered at a pH of 6-8 andcontaining 5-20 percent by volume of. an alcohol, selected from theclass consisting of methanol and ethanol and mixtures thereof and 4-6percent by weight of an alkali metal salt of an amino acid having notmore than six carbon atoms, stirring and centrifuging the mixture,filtering the supernatant and dialyzing the filtered supernatant againstdistilled water, and lyophilizing the dialyzed supernatant.

47 A substance capable of increasing the activity of thromboplasticmaterial prepared from prothrombin-free plasma, selected from the classconsisting of horse, bovine, human, and rabbit prothrombin-free plasma,by adding, at a temperature of 0 to -5 C., to a unit volume of theplasma, maintained at a temperature of -5 to 10 C.

about four volumes of a first aqueous alcohol solution buffered at a pHof 4.2-4.6 and containing about 25 percent by volume of an alcoholselected from the class consisting of methanol and ethanol and mixturesthereof, stirring and subsequently centrifuging the mixture while thetemperature thereof is maintained at 0 to 5 C., separating thesupernatant from the residue; and adding to the residue while thetemperature is 0 to 5 C., 3-5 volumes based-on plasma volume of a secondaqueous alcohol solution, at a temperature of 0 to 5 C., buffered at apH of 6.8-7.2 and containing about 16 percent by volume of an alcoholselected from the class consisting of methanol and ethanol and mixturesthereof, and 5.4-5.8 percent by weight of an alkali metal salt of anamino acid having not more than six carbon atoms, stirring andsubsequently centrifuging while thetemperature is at 0 to 5 C.,filtering and dialyzing the filtered supernatant against the distilledwater while the temperature is at 15 C., and lyophilizing the clialyzedsupernatant.

References Cited in the file of this patent UNITED STATES PATENTS2,162,863 Ripke June 20, 1939 OTHER REFERENCES Hardy: Chem. Abst., vol.45, March 1951, p. 2046a.

Cohn: Ann. Int. Med., vol. 26, No. 3, March 1947, pp. 341-352 (pp. 346,347, 349-352 relied on).

Surgenor et al.: I. A. C. S., vol. 74, No. 13, July 5, 1952, pp.3448-3450.

Tullis: Blood Cells and Plasma Proteins, 1953, Academic Press, Inc., N.Y. C., p. 36.

Edsall: Advances in Protein Chem, vol. 3, 1947, Academic Press, Inc., N.Y. C., pp. 440-445.

UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION Patent No2,847,348 August 12, 1958 Heron O, Singher et a].

It is hereby certified that error appears in the printed specificationof the above numbered patent requiring aerreotien and that the saidLetters Patent should read as corrected telew- Column 4, line 58, for"656 ml," read 565 ml, column 6, line '71, strike out "While the" andinsert instead at a lines '71 and '72, strike out "at a" and insertinstead While the Signed and sealed this 28th day'of October 1958,

(SEAL Attesn KARL Ho AKIN-E ROBERT c. WATSON Attesting OflicerCommissioner of Patents

1. A PROCESS FOR THE PREPARATION OF A SUBSTANCE CAPABLE OF INCREASINGTHE ACTIVITY OF THROMBOPLASTIC MATERIAL, CONDUCTED AT LOW TEMPERATURETHROUGHOUTY, COMPRISING THE STEPS: ADDING TO A UNIT COLUME OFPROTHROMBIN-FREE PLASMA SELECTED FROM THE CLASS CONSISTING OF HORSE,BOVINE, HUMAN, AND RABBIT PROTHROMBIN-FREE PLASMA, THREE TO FIVE COLUMESOF A FIRST AQUEOUS ALCOHOL SOLUTIONA BUFFERED AT A PH OF 4-5 ANDCONTAINING ABOUT 15-30 PERCENT BY VOLUME OF AN ALCOHOL SELECTED FROM THECLASS CONSISTING OF METHANOL AND ETHANOL AND MIXTURES THEREOF, STIRRUNGAND CENTRIFUGUNG THE MIXTURE, SEPARATING THE SUPERNATANT FROM THERESIDUE; AND ADDING TO THE RESIDUE THREE TO FIVE VOLUMES BASED ONBPLASMA VOLUME OF A SECOND AQUEOUS ALCOHOL SOLUTION BUFFERED AT A PH OF6-8 AND CONTAINING 5-20 PRECENT BY VOLUME OF AN ALCOHOL SELECTED FROMTHE CLASS CONSISTING OF METHANOL AND ETHANOL AND MIXTURES THEREOF AND4-6 PERCENT BY WEIGHT OF AN ALKALI METAL SALT OF AN AMINO ACID HAVINGNOT MORE THAN SIX CARBON ATOMS, STIRRING AND CETRIFUGING THE MIXTURE,FILTERING THE SUPERNATANT, DIALYZING THE FILTERED SUPERANATANT AGAINSTDISTILLED WATER, AND LYOPHILIZING THE DIALYZED SUPERNATANT.